Introduction of Stat3 Pathway.
Stat3 is a member of the Stat family which are latent transcription factors activated in response to cytokines/growth factors to promote proliferation, survival, and other biological processes. Stat3 is activated by phosphorylation of a critical tyrosine residue mediated by growth factor receptor tyrosine kinases, Janus kinases, and/or the Src family kinases, etc. These kinases include but not limited to EGFR, JAKs, Abl, KDR, c-Met, Src, and Her2 [1]. Upon tyrosine phosphorylation, Stat3 forms homo-dimers and translocates to the nucleus, binds to specific DNA-response elements in the promoters of the target genes, and induces gene expression [2].
Importance of Stat3 Pathway in Targeting Conventional Aspects of Cancers.
In normal cells, Stat3 activation is transient and tightly regulated, lasting from 30 minutes to several hours. However, Stat3 is found to be aberrantly active in a wide variety of human cancers, including all the major carcinomas as well as some hematologic tumors. Stat3 plays multiple roles in cancer progression. As a potent transcription regulator, it targets genes involved in cell cycle, cell survival, oncogenesis, tumor invasion, and metastasis, such as Bcl-xl, c-Myc, cyclin D1, Vegf, MMP-2, and survivin [3-8]. It is also a key negative regulator of tumor immune surveillance and immune cell recruitment [9-11].
Ablating Stat3 signaling by antisense, siRNA, dominant-negative form of Stat3, and/or blockade of tyrosine kinases causes cancer cell-growth arrest, apotosis, and reduction of metastasis frequency in vitro and/or in vivo [2, 4, 12, 13].
Importance of Stat3 Pathway in Other Diseases.
Activation of Stat3 by various cytokines, such as Interleukin 6 (IL6) has been demonstrated in a number of autoimmune and inflammatory diseases. Recently, it has been revealed that the Stat3 pathway promotes pathologic immune responses through its essential role in generating TH17 T cell responses [14]. In addition, Stat3 pathway mediated inflammation is the common causative origin for atherosclerosis, peripheral vascular disease, coronary artery disease, hypertension, osteroprorosis, type 2 diabetes, and dementia. Therefore, Stat3 inhibitors may be used to prevent and treat autoimmune and inflammatory diseases as well as the other diseases listed above that are caused by inflammation.
Introduction of Cancer Stem Cells (CSCs).
Cancer stem cells (CSCs) are a sub-population of cancer cells (found within tumors or hematological cancers) that possess characteristics normally associated with stem cells. These cells are tumorigenic (tumor-forming), in contrast to the bulk of cancer cells, which are non-tumorigenic. In human acute myeloid leukemia the frequency of these cells is less than 1 in 10,000 [15]. There is mounting evidence that such cells exist in almost all tumor types. However, as cancer cell lines are selected from a sub-population of cancer cells that are specifically adapted to growth in tissue culture, the biological and functional properties of these cell lines can change dramatically. Therefore, not all cancer cell lines contain cancer stem cells.
CSCs have stem cell properties such as self-renewal and the ability to differentiate into multiple cell types. They persist in tumors as a distinct population and they give rise to the differentiated cells that form the bulk of the tumor mass and phenotypically characterize the disease. CSCs have been demonstrated to be fundamentally responsible for carcinogenesis, cancer metastasis, and cancer reoccurrence. CSCs are also often called tumor initiating cells, cancer stem-like cells, stem-like cancer cells, highly tumorigenic cells, or super malignant cells.
Clinical Implications of Cancer Stem Cells.
The existence of cancer stem cells has several implications in terms of cancer treatment and therapy. These include disease identification, selective drug targets, prevention of cancer metastasis and recurrence, treatment of cancer refractory to chemotherapy and/or radiotherapy, treatment of cancers inherently resistant to chemotherapy or radiotherapy and development of new strategies in fighting cancer.
The efficacy of cancer treatments are, in the initial stages of testing, often measured by the amount of tumor mass they kill off. As CSCs would form a very small proportion of the tumor and have markedly different biologic characteristics than their differentiated progeny, the measurement of tumor mass may not necessarily select for drugs that act specifically on the stem cells. In fact, cancer stem cells are radio-resistant and also refractory to chemotherapeutic and targeted drugs. Normal somatic stem cells are naturally resistant to chemotherapeutic agents—they have various pumps (such as MDR) that efflux drugs, higher DNA repair capability, and have a slow rate of cell turnover (chemotherapeutic agents naturally target rapidly replicating cells). Cancer stem cells, being the mutated counterparts of normal stem cells, may also have similar functions which allow them to survive therapy. In other words, conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor that are unable to generate new cells. A population of cancer stem cells which gave rise to it could remain untouched and cause a relapse of the disease. Furthermore, treatment with chemotherapeutic agents may only leave chemotherapy-resistant cancer stem cells, so that the ensuing tumor will most likely also be resistant to chemotherapy. Cancer stem cells have also been demonstrated to be resistant to radiotherapy (XRT) [16, 17].
Since surviving cancer stem cells can repopulate the tumor and cause relapse, it would be possible to treat patients with aggressive, non-resectable tumors and refractory or recurrent cancers, as well as prevent the tumor metastasis and recurrence by selectively targeting cancer stem cells. Development of specific therapies targeted at cancer stem cells therefore holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. The key to unlocking this untapped potential is the identification and validation of pathways that are selectively important for cancer stem cell self-renewal and survival. Though multiple pathways underlying tumorigenesis in cancer and in embryonic stem cells or adult stem cells have been elucidated in the past, no pathways have been reported for cancer stem cell self-renewal and survival.
Identification and Isolation of CSCs.
The methods on identification and isolation of cancer stem cells have been reported. The methods are used mainly to exploit the ability of CSCs to efflux drugs, or are based on the expression of surface markers associated with cancer stem cells.
CSCs are resistant to many chemotherapeutic agents, therefore it is not surprising that CSCs almost ubiquitously overexpress drug efflux pumps such as ABCG2 (BCRP-1) [18-22], and other ATP binding cassette (ABC) superfamily members [23, 24]. The side population (SP) technique, originally used to enrich hematopoetic and leukemic stem cells, was first employed to identify CSCs in the C6 glioma cell line [25]. This method, first described by Goodell et al., takes advantage of differential ABC transporter-dependent efflux of the fluorescent dye Hoechst 33342 to define a cell population enriched in CSCs [21, 26]. The SP is revealed by blocking drug efflux with verapamil, so that the SP is lost upon verapamil addition.
Efforts have also focused on finding specific markers that distinguish cancer stem cells from the bulk of the tumor. Markers originally associated with normal adult stem cells have been found to also mark cancer stem cells and co-segregate with the enhanced tumorigenicity of CSCs. The most commonly expressed surface markers by the cancer stem cells include CD44, CD133, and CD166 [27-33]. Sorting tumor cells based primarily upon the differential expression of these surface marker(s) have accounted for the majority of the highly tumorigenic CSCs described to date. Therefore, these surface markers are well validated for identification and isolation of cancer stem cells from the cancer cell lines and from the bulk of tumor tissues.